Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

This method allows the study of proton transport activity of respiratory membrane enzymes like cytochrome BO3 in a natural lipid bi-layer environment. The main advantages of this single enzyme technique is the possibility to start and stop enzymatic reactions at any moment using electrochemistry. Using a glass syringe transfer 200 microliters of chloroform stock of lipid polar extract from Escherichia coli into glass vials to make five milligram aliquots.

Add 50 microliters of one milligram per milliliter ubiquinone 10 to the lipids to make the final ratio of one to 100 ubiquinone 10 to lipids. To the mixture add 20 microliters of one milligram per milliliter long wave length fluorescent dye labeled lipid abbreviated FDLL. Homogenize the chloroform solution by short vortexing and evaporate most of the chloroform under a gentle nitrogen or argon flow.

Remove the chloroform traces entirely by further evaporation under vacuum for at least one hour. Add 312.5 microliters of 40 millimolar MOPS pH 7.4 buffer to one aliquot of lipids ubiquinone 10 FDLL dry mix. Mix the vortex til the lipid film is fully re-suspended followed by two minutes of treatment in an ultrasonic bath.

Next add 125 microliters of 25 millimolar HPTS, a pH sensitive fluorescent dye that will be encapsulated inside the liposomes. Now add 137.5 microliters of 250 millimolar OGP surfactant. After mixing using vortex, sonicate the mixture in an ultrasonic water bath for 10 minutes to ensure all lipids are solubilized into surfactant micelles.

Then transfer the dispersion into a 1.5 milliliter plastic tube. Add the required amount of cytochrome BO3 and add ultra pure water to make a total volume of 50 microliters. Incubate the reconstitution mixture at four degrees celsius for 10 minutes on a roller mixer.

Next weigh 50 milligrams of polystyrene microbeads into each of the two 1.5 milliliter tube caps. Then weigh 100 milligrams of polystyrene microbeads into another two 1.5 milliliter tube caps. Close the caps with paraffin film to prevent drying.

Add the first 50 milligrams of polystyrene microbeads into the reconstitution mixture by putting the cap with polystyrene microbeads on the 1.5 milliliter plastic tube with the prepared dispersion. Then perform a short spin for a couple of seconds to bring microbeads to the bottom of the tube. Incubate the suspension at four degrees celsius on a roller mixer to allow the polystyrene microbeads to absorb the surfactant for 30 minutes.

Repeat the additions of polystyrene microbeads and incubations as follows. Add 50 milligram of microbeads for 60 minutes of incubation. Then add 100 milligram of microbeads for 60 minutes of incubation.

And finally, add 100 milligram of microbeads for 120 minutes of incubation. Separate the proteoliposome solution from the polystyrene microbeads using a micropipette with a thin tip. Dilute the dispersion in 90 milliliters of MOPS buffer in TI 45 ultracentrifuge tube.

Ultracentrifuge the dispersion using a type 45 TI rotor at 125, 000 times G for one hour to pellet the proteoliposomes. Following ultracentrifugation, discard the supernatant and rinse the pellets with MOPS buffer without resuspending the pellet. After discarding the rinsing buffer, re-suspend the proteoliposomes in 500 microliters of MOPS buffer by pipetting it back and forth with a thin tipped micropipette before transferring the suspension to a 1.5 milliliter plastic tube.

Following centrifugation for five minutes at 12, 000 times G to remove the debris, transfer the supernatant which is the reconstituted proteoliposomes into a new vial. Glue up to nine glass cover slips onto an evaporated gold surface using bi-component low-fluorescence epoxy. Cure the glue at 80 degrees celsius for four hours.

Just before modification with a self-assembled mono-layer, detach the glass cover slips from the silicone wafers with a blade. Due to the thinness of the cover slips, take care when detaching the cover slips to not crack or break the glass slides. Dip the freshly detached gold-coated cover slips into a 6-marcaptohexanol solution and leave at 20 to 25 degrees celsius overnight to form the self assembled mono-layer.

The next day, remove the gold-coated cover slips from the solution and wash briefly with water or methanol and then with isopropanol. Dry the gold-coated cover slips under a gentle gas flow. Assemble the gold-coated cover slip in a spectro-electrochemical cell as diagrammed in the text protocol.

Make contact to the gold with a flat wire outside an area defined by a rubber O-ring and tightly screw down the electrochemical cell on the top of it. Add two milliliters of the electrolyte buffer solution and place the reference and auxiliary electrodes in the cell. Proceed to check the quality of the self-assembled mono-layer and run blanks as described in the text protocol.

Add proteoliposomes to the electrochemical cell at 0.5 milligrams per milliliter final lipids concentration and mix slightly with a pipette. Wait 30 to 60 minutes at room temperature until the absorption of proteoliposomes on the electrode surface is finished. Wash the cell by changing the buffer solution at least 10 times, but avoid leaving the electrode surface completely dry.

Now, run electrochemical impedance spectroscopy at open cell potential to confirm the self-assembled mono-layer on the gold electrode remains unchanged. Run cyclic voltammograms with scan rates 10 and 100 millivolts per second to observe catalatic ubiquinol oxidation and oxygen reduction by cytochrome BO3 at onset potentials of electrochemical quinone reduction. Modify the gold electrode as before but using five micrograms per milliliter proteoliposomes.

For single enzyme studies reduce the cytochrome BO3 to lipid ratio to between 0.1 and 0.2%Place a drop of immersion oil and then the electrochemical cell on the 60 times oil objective of an inverted fluorescence microscope. Using appropriate filters for FDLL fluorescence, focus on the electrode surface. Single liposomes should appear as bright spots at the diffraction limit of the microscope objective.

Take an image of FDLL florescence. Switch to the one of HPTS florescence filter sets on the microscope to verify that HTPS florescence is clearly visible and distinguishable from the background. Increase the light intensity if it is not the case.

Program the microscope software to perform a timed image acquisition by alternating two HTPS filter sets. To do so, set a one second exposure then navigate to menu applications. Then Define/Run ND Acquisition and select two HTPS channels.

Set the delay between image acquisitions at minimum. In this experiment use 0.3 second delay and five minute total duration. Adjust the settings of the potentiostat to change the potential during the image acquisition as indicated in the text protocol.

Now, simultaneously run the timed image acquisition on the microscope and the potential sequence on the potentiostat by manually starting both measurements at the same time. Shown here are fluorescence images of liposomes absorbed on the electrodes at three different coverages. The dye containing liposomes are visible on the images as bright spots.

The central part of the image was photo-bleached to reveal the background florescence level. The images on two HTPS channels are super imposable where the ratio between the two channels corresponds to a pH of 7.4 used in this experiment. A larger number of liposomes are visible with the FDLL channel, which indicates the presence of liposomes that have no HPTS encapsulated.

The difference between the HPTS and FDLL channels is more pronounced at higher coverages. Possibly because at high liposome coverage, liposomes are more likely to burst or fuse on the surface. Here, the change of a liposome florescence on two HTPS channels is shown as a 3D surface plot of the corresponding area during 300 seconds of the experiment.

The reductive potential was applied between 60 seconds and 180 seconds. The corresponding plot of volume metric intensity ratio of two HPTS channels versus time is shown. Shown here are the medians of all vesicle pH changes within a single image when cytochrome BO3 content is 1.3%An increase in pH is clearly visible when a potential between 0.1 and 0.3 volts is applied.

When cytochrome BO3 content is a much lower value of 0.1%the median curves become almost indistinguishable from those of empty liposomes. It is important to ensure complete removal of the surfactant from the liposome dispensations since the residuals may increase the permeability of the liposomes to protons. These methods allow us to ask specific questions on the natural lipophilic quinone substrate in contrast to the often used water-soluble quinone analogues.

Further more, the single enzyme experiments identified a previously unknown leak state where the protein acts as a proton channel where protons freely flow backwards.